1) Field of the Invention
This invention relates to signal generating systems for specific binding assays. More particularly, it relates to the enhancement of available signal by reduction in self-quenching of signal moieties associated with the assay systems.
2) Brief Description of the Prior Art
The development of specific binding assay techniques has provided extremely useful analytical methods for determining various substances of diagnostic, medical, environmental and industrial importance which appear in liquid media at very low concentrations. Specific binding assays are based on the specific interaction between a bindable analyte under determination, and a binding partner therefor, i.e., analyte-specific moiety. The binding of the analyte-specific moiety and interaction of any additional reagents, if necessary, effect a mechanical separation of bound and unbound labeled analyte or affect the label in such a way as to modulate the detectable signal. The former situation is normally referred to as heterogeneous and the latter as homogeneous, in that the latter technique does not require a separation step. Where one of the analyte and its binding partner is a hapten or antigen and the other is a corresponding antibody, the assay is known as an immunoassay. See, generally, Odell and Daughaday (Eds.), Principles of Competitive Protein-Binding Assays, J. B. Lippincott Co., Philadelphia (1971). Where one of the analyte and its binding partner is a target nucleic acid sequence and the other is a complementary nucleic acid sequence, the assay is known as a nucleic acid hybridization assay. See, generally, Falkow, et.al., U.S. Pat. No.4,358,535, Kourilsky, et. al., U.S. Pat. No. 4,581,333, Albarella, et. al., U.S. Pat. No. 4,563,417 and Paau, et. al. U.S. Pat. No. 4,556,643. In conventional label conjugate specific binding assay techniques, a sample of the liquid medium to be assayed is combined with various reagent compositions. Such compositions include a label conjugate comprising a binding component incorporated with a label. The binding component in the conjugate participates with other constituents, if any, of the reagent composition and the ligand in the medium under assay to form a binding reaction system producing two species or forms of the conjugate, e.g., a bound-species (conjugate complex) and a free-species. In the bound-species, the binding component of the conjugate is bound by a corresponding binding partner whereas in the free species, the binding component is not so bound. The amount or proportion of the conjugate that results in the bound species compared to the free species is a function of the presence (or amount) of the analyte to be detected in the test sample. An alternative format for specific binding assays is the "sandwich" or "capture" assay in which the target analyte is bound between a first specific binding partner, which can be directly or indirectly fixed to a solid matrix, and a second specific binding partner, which is associated with a signal generating or labeling system.
Early hybridization techniques involved the use of radioactive labels such as H.sup.3, P.sup.32, or I.sup.123. An alternative is disclosed in British Patent No. 2,019,408 which describes polynucleotide probes labeled with biotin through cytochrome C linkage groups. Bound probes are detectable by enzyme-labeled avidin through a chromogenic reagent signalling system. An improved approach to labeling probes with low molecular weight analytes, such as biotin, is described in European Patent Application No. 63,879. In this technique, 5-allylamine-deoxyuridine triphosphate (dUTP) derivatives are condensed with the desired analyte label in a manner which is not disruptive of hybridization. The thus modified nucleotide is incorporated by standard enzymatic methods into the desired oligo- or polynucleotide probe. The use of light emitting labels is reported in European Patent Application Nos. 70,685 and 70,687. Other representative patent literature pertaining to hybridization assays includes U.S. Pat. Nos. 4,302,204 concerning the use of certain water soluble polysaccharides to accelerate hybridization on a solid phase; 4,358,535 concerning the detection of pathogens in clinical samples; and 4,395,486 concerning the detection of the sickle cell anemia trait using a synthetic oligonucleotide probe.
Labeling systems for protein binding or immunoassay type specific binding assay systems are numerous and well known in the art. A review of many such labeling systems is presented by U.S. Pat. Nos. 4,134,792; 4,213,893; 4,230,797; 4,238,195; 4,318,980; 4,318,981; 4,318,982; 4,442,204, 4,492,751 and EPO 140,521.
Visor, et. al., J. Pharm. Sci., 70:469(1981) also provides an overview and literature survey regarding fluorescent immunoassay. Another of many review articles published is Soini, et.al., Clin. Chem., 25:353(1979). Exley, et.al., J. Steroid Biochem., 14:1297(1981) reports an immunoassay using a poly-L-lysine carrier to which are attached multiple fluoresceins. Attachment of fluorescein to oligonucleotides has also been reported. See Richardson, et.al., Nuc. Acids Res., 11:6167(1983) and the literature cited therein. Various refinements and improvements of this basic fluorescent specific binding assay technology have evolved.
Fluorescent binding assays of the homogeneous type have been reported which avoid the usually disadvantageous separation step. One such method is based on quenching or enhancement of fluorescence upon binding of a fluorescer-ligand conjugate with its target binding partner(analyte). Examples are provided in Belgian Patent No. 858,722 and German Offenlegungsschriften Nos. 2,716,276 and 2,716,515. A variation of this assay method is described in U.S. Pat. No. 3,996,345 which employs a specific quenching substance as a counterpart to the fluorescer label. These methods are reviewed in Visor, et. al. and Soini, et. al., supra.
Hemmilia, Clin. Chem. 31:359(1985) provides a more recent review of fluoroimmunoassays and immunofluorimetric assays. It notes that the close proximity of two fluorescent probes in a protein can cause self-quenching if their absorption and emission spectra overlap. It also states that despite their short Stokes' shift and consequent sensitivity to concentration quenching, polymeric fluorescein labels have also been used. Hassan, et. al., FEBS Letters 103:339(1979) is cited. Also mentioned is that self-quenching can be eliminated by introducing releasable linkages to polymeric labels. The quenched, polymer-bound probes are hydrolyzed in a solution of monomeric fluorescent dye once the immunoreaction is complete. Yaverbaum, et. al., U.S. Pat. No. 4,576,912 is cited.
Hassan, et. al., supra, addresses the "concentration quenching" of fluorescein in multi-substituted polymers. As illustrated in FIG. 1 thereof, an excess of anti-fluorescein antibodies is added to the multi-fluorescein substituted polymer. The remaining unoccupied antibody sites are "back-titrated" by addition of a monomeric fluorescein reagent. The monomeric fluorophores which become antibody-bound are also efficiently quenched, while the remander give a fluorescent signal related to the number of fluoresceins on the polymer but unquenched because the fluorescence is produced by these conventional monomeric units in solution and not by those of the multi-fluoresceinated polymer. In fact, complete quenching, intentionally, of the multi-fluoresceinated polymer is the result.
Thus, the disadvantageous characteristic of fluorescent molecules to emit diminished signal when in high concentrations or close proximity has been addressed by resort to alternative label systems or conversion of multifluoresceinated label systems to ones in which signal is read from conventional fluorescein polymers.